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本研究探讨树突状细胞(DC)吞噬经PUVA(8-methoxypsoralen plus UVA irradiation,8-甲氧基补骨脂素联合长波紫外线照射的体外光化学治疗)处理的同种异基因淋巴细胞后对于同基因T细胞的免疫调控作用。分离LEW大鼠骨髓细胞,经IL-4和GM-CSF共同诱导制备骨髓来源的LEW大鼠DC。分离DA大鼠脾淋巴细胞(SP),制备经PUVA处理的DA大鼠脾淋巴细胞(PUVA-SP),检测PUVA-SP的凋亡率。在体外将PUVA-SP或SP与受者骨髓来源的未成熟DC共同培养,检测上述处理对受者DC表面分子CD40、CD86、MHC-Ⅱ的影响。以CFSE标记PUVA-SP,通过荧光显微镜观察LEW大鼠DC吞噬DA大鼠PUVA-SP的情况。通过混合淋巴细胞培养(MLR),检测吞噬DA大鼠PUVA-SP的LEW大鼠DC(PUVA-SPDC)对LEW大鼠T细胞的刺激作用,同时检测MLR培养上清中细胞因子IL-4、IL-10、IL-2、IFN-γ的浓度。结果表明:PUVA处理能够在24小时内诱导62.87%的DA大鼠脾淋巴细胞发生早期凋亡。LEW大鼠DC与DA大鼠PUVA-SP共培养后,可有效吞噬PUVA-SP。LEW大鼠DC与DA大鼠SP混合培养后,其表面分子MHC-Ⅱ、CD40以及CD86的表达阳性率分别为65.6%、45.6%和36.5%,明显高于未经处理的受者DC组27.7%、22.9%和13.0%(p<0.01);而DC与PUVA-SP混合培养后,其表面分子MHC-Ⅱ、CD40及CD86的表达仍保持较低的水平,分别为25.7%、21.0%和13.4%,与未经任何处理的对照组DC相当(p>0.05),而远远低于LPS刺激后的成熟DC(82.3%、71.7%和63.2%)(p<0.01)。DA大鼠SP共培养后的LEW大鼠DC可以刺激LEW大鼠T细胞显著增殖,刺激指数在加载DC数量为2×103、10×103、20×103组分别为1.7546、3.9798和5.0220,而吞噬DA大鼠PUVA-SP的LEW大鼠DC则诱导了T淋巴细胞低反应性,刺激指数分别为1.0120、1.1518和1.4093,二者差异有统计学意义(p<0.01)。此外,与SPDC相比,PUVA-SPDC明显抑制了T细胞IFN-γ的分泌(140.17±2.28vs37.67±1.76)(p<0.01),刺激了IL-10(33.11±1.82vs69.58±1.83)(p<0.01)、IL-4的分泌(11.11±1.07vs23.83±0.73)(p<0.01),但对IL-2的分泌没有抑制作用(418.18±4.55vs423.86±5.39)(p>0.05)。结论:PUVA-SPDC能够诱导大鼠T细胞对异基因抗原的免疫低反应,还可在体外诱导LEW大鼠T淋巴细胞由Thl向Th2免疫偏移。
This study investigated the effect of dendritic cells (DC) phagocytosing allogeneic lymphocytes treated with PUVA (8-methoxypsoralen plus UVA irradiation in vitro photochemical therapy with 8-methoxy psoralen combined with UVB radiation) Gene T cell immune regulation. LEW rat bone marrow cells were isolated and induced by bone marrow-derived LEW rat DCs induced by IL-4 and GM-CSF. Splenic lymphocytes (SPs) of DA rats were isolated and splenic lymphocytes (PUVA-SP) of PUVA-treated DA rats were prepared to detect the apoptosis rate of PUVA-SP. PUVA-SP or SP was co-cultured with recipient bone marrow-derived immature DCs in vitro to examine the effects of the above treatments on the DC surface molecules CD40, CD86 and MHC-II. The CFU-labeled PUVA-SP was used to observe the phagocytosis of DA rats PUVA-SP by LEW rat DC by fluorescence microscopy. The stimulation effect of LUVA (PUVA-SPDC) phagocytosed DA PUVA-SP on T cells of LEW rats was detected by mixed lymphocyte culture (MLR), and the cytokines IL-4, IL-10, IL-2, IFN-γ concentrations. The results showed that PUVA treatment could induce early apoptosis of splenic lymphocytes in 62.87% DA rats within 24 hours. PUVA-SP can be effectively phagocytosed by LEW rat DC co-cultured with PUVA-SP of DA rats. The positive expression rates of MHC-Ⅱ, CD40 and CD86 on LEW rat DC and DA rat SP were 65.6%, 45.6% and 36.5%, respectively, which were significantly higher than those of the untreated recipient DC group 27.7 %, 22.9% and 13.0%, respectively (p <0.01). However, the expression of MHC-Ⅱ, CD40 and CD86 on the surface of DCs incubated with PUVA-SP remained low at 25.7%, 21.0% and 13.4%, which was significantly lower than that of control group without any treatment (p> 0.05), but far lower than that of mature DCs (82.3%, 71.7% and 63.2%) after LPS stimulation (p <0.01). DCs of LEW rats co-cultured with DA rats could significantly stimulate the proliferation of T lymphocytes in LEW rats. The stimulation index was 1.7546, 3.79798 and 5.0220 respectively when the loading DCs were 2 × 103, 10 × 103 and 20 × 103, respectively LEW rats with phagocytosis of DA rats PUVA-SP induced a low reactivity of T lymphocytes with stimulation indices of 1.0120, 1.1518 and 1.4093, respectively, with significant difference (p <0.01). In addition, PUVA-SPDC significantly inhibited the secretion of IFN-γ by T cells (140.17 ± 2.28 vs 37.67 ± 1.76) (p <0.01) and stimulated IL-10 (33.11 ± 1.82 vs 69.58 ± 1.83 (p <0.01), IL-4 secretion (11.11 ± 1.07 vs23.83 ± 0.73) (p <0.01), but had no inhibitory effect on IL-2 secretion (418.18 ± 4.55 vs423.86 ± 5.39) > 0.05). CONCLUSION: PUVA-SPDC can induce immunoreactivity in rat T cells against allogeneic antigens and induce immunostains from Th1 to Th2 in T lymphocytes of LEW rats induced in vitro.